Apparatus and method for the preparation of fluid bearing materials for surface analysis in a vacuum

ABSTRACT

An apparatus and method for the presentation of organic samples at low temperature and under vacuum for analysis using a surface analysis technique is described. The apparatus carries a sample trapped between two hinged plates which is subsequently cryogenically-cooled and then inserted into a vacuum system where it locates on a cold stage. The device has a mechanism which is used to fracture the sample whilst under vacuum and open the two halves of the sample ready for analysis. It thus provides a reliable automated method of freeze-fracture, replacing current unreliable manual methods.

FIELD OF THE INVENTION

This invention relates to an apparatus and method that facilitates the surface and near-surface analysis of fluid bearing materials such as biological and organic materials where the fluid content needs to be frozen to prevent evaporation of the fluid in the vacuum system of the analyser prior to, during and after the analysis process. Surface ice formation and surface contamination is also minimised.

BACKGROUND OF THE INVENTION

A variety of surface and near surface analysis techniques are widely used to identify chemical structure and composition on or just below the surface in solid inorganic materials. These techniques are typically highly dependent on surface condition and cleanliness and so the condition of the surface of sample to be analysed is critical to obtaining optimal results.

Typically, a surface analytical technique comprise of a probe beam or primary radiation comprising charged particles, neutral particles or photons which acts so as to stimulate the surface or near surface to emit a secondary emission or radiation in the form of charged particles, neutral particles or electromagnetic radiation of which the mass, energy, wavelength, intensity, dispersion or any combination thereof gives a measure of a quantifiable parameter of the sample. Such techniques can include Secondary Ion Mass Spectrometry (SIMS), in which the sample is bombarded by a beam of ions to dislodge secondary ions from the surface, X-ray Photoelectron Spectroscopy (XPS), in which electrons are ejected from a sample by incident x-rays and measurement is made of the electrons' energy spectra, and Auger Electron Spectroscopy (AES), in which measurement is made of Auger electrons' energy spectra.

Because of the surface specificity of surface analytical techniques, it is usually a requirement that the analysis process takes place in an Ultra High Vacuum (UHV) environment, normally in the range of 1 e-6 mbar to 1 e-11 mbar, to prevent previously cleaned surfaces from being contaminated on the surface by gas absorption and particulates. The analysis of many solid materials such as most metals, alloys, polymers, ceramics and semiconductors is now relatively straightforward as the samples can be surface cleaned by a number of established techniques. This cleaning of the sample takes place either prior to the sample being placed in the vacuum, under vacuum in the analysis chamber or whilst in a secondary or sample preparation chamber under vacuum which is attached to the high vacuum analysis chamber. Such sample-preparation techniques are well known, are accepted practice for most solid materials, are well understood and relatively inexpensive to implement. Additionally, solid samples typically do not significantly outgas during the analysis, a process which can lead to the degradation of the sample and to contamination of the analysis chamber.

More recently, a great deal of interest has been shown in the analysis of biological samples, which typically have a relatively high fluid content, by the aforementioned surface analysis techniques, for studying the chemical composition of cells and the mapping the concentrations of these chemicals inside the cell in order to understand functionality. As a result, a great deal of effort has been made to find methods and techniques to undertake this type of analysis. The SIMS technique, in particular, has seen advances in the past few years which extend the potential of the technique from mere elemental identification into extensive imaging and chemical mapping of complex organic surfaces in both 2 and 3 dimensions (for example: Fletcher et al; Molecular depth profiling of organic and biological materials and Vickerman; Static SIMS—A Surface Mass Spectrometry for Organic Materials in Secondary Ion Mass Spectrometry).

In SIMS, a primary ion beam is incident on the sample surface and primary ion impacts cause secondary ions to be ejected from the surface. These can be captured and analysed in a mass spectrometer. With scanning of the primary beam, data from the mass spectrometer can be related to specific areas (pixels) on the surface to provide a mass spectrum for each pixel position creating a chemical image or map of the surface. Using atomic primary ions, it is very rare to see whole molecules or large fragments in the spectra, however, advances in cluster ion beam technology have substantially increased the mass range that can be measured using the SIMS technique and also the sensitivity of that measurement.

The use of cluster primary ions, such as fullerene molecular ions (e.g. C₆₀ ⁺), has made SIMS applicable to the analysis of biological and biochemical samples, subject to overcoming two problems in sample preparation: firstly, that of stopping vaporisation and outgassing into the vacuum system of the analysis instrument, and secondly, that of presenting a clean surface for analysis.

Unlike solid samples, biological samples in their native state are typically formed of at least 70% liquid which makes them not particularly conducive to analysis in vacuum systems due to the problems of evaporation or outgassing.

Outgassing is a process whereby the liquid, vapour or gas contained in a sample effectively evaporates form the sample at a highly accelerated rate when placed in a vacuum. This outgassing results in both changes to the structure, the chemical composition of the sample and can have a detrimental effect on the vacuum quality in the analyser making analysis difficult or almost impossible. In order to overcome these problems, the samples may be converted to a relatively rigid and solid form by rapidly freezing them. This greatly reduces the outgassing, provides a fixed surface and structure facilitating structurally related analysis and retains the chemical information thus making SIMS analysis a very powerful and practical tool.

Rapid freezing of samples for microscopy has been in use since well before the 1960's but the vast majority of analysis was carried out in near normal or normal atmospheric conditions. This rapid freezing is usually achieved using liquid nitrogen which has a boiling point of 77K. At these low temperatures, ice formation on the surface has always been a problem, but a simple cleaning by suction removal of ice crystals formed on the surface has often sufficed to make the technique viable for early analytical purposes. Where a cleaner surface is required, the sample is frozen between two microscope slides and then, once frozen, the slides are forcibly separated. This results in the sample being fractured along the path of least resistance which is hopefully through the sample and reveals features of interest for analysis.

The resultant surface exposed is clean and highly suited for surface analysis. This technique is known as freeze-fracturing. Alternatively, the sample can be cleaved by a blade exposing a clean surface, a process known as freeze-cleaving. A variety of specific techniques have been developed for optical microscopy, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) (for example, Nakamoto et al.; In-Situ Observation of Freeze Fractured Red Blood Cell with High-Vacuum Low-Temperature Atomic Force Microscope).

With the development of powerful surface analytical techniques such as imaging and mapping SIMS and the growing interest in performing surface analysis on organic samples, simple adaptations of the freeze-fracture and freeze cleave techniques (for example as described by Moller at al.; Introduction of a cryosectioning ToF SIMS instrument for analysis of non-dehydrated biological samples), have been made to permit their use in vacuum systems. However, the surface cleanliness and vacuum required to carry out a surface analysis imposes a unique set of challenges not previously encountered.

The following description uses a SIMS instrument but can easily be extrapolated to any surface analytical technique requiring HV or UHV.

The primary problem encountered is that after freeze fracturing or freeze cleaving, any exposure of the frozen sample surface to the normal atmosphere leads to the creation of a thin layer of ice crystals on the surface in only a few seconds thereby rendering the sample effectively unusable for surface analysis.

One method is to assemble the frozen slides with the sample placed between them, mount them securely to a sample holder used in the SIMS analyser, perform a simple fracture in a dry gas environment and then rapidly transfer the sample to the insertion lock and onto a cold stage where the frozen condition is hopefully preserved. However, even dry gas contains sufficient moisture to cause the formation of ice crystals on the surface and the whole process is highly dependent on the operator's manual dexterity and speed in loading the sample into the vacuum to prevent the sample warming or excessive ice forming on the surface. In either case it is not suitable for surface analysis.

Alternatively, the frozen slide & sample assemblage is transferred to a cold stage in the instrument via an insertion lock and brought under vacuum. Once under vacuum, the slides can be prized apart manually using a movable probe (vacuum probe) through the vacuum wall, fracturing the sample. All of this must take place without any frosting of the sample or sample holder whatsoever. At the end of this procedure, the lower glass slide should remain in place with a new sample surface exposed for analysis. There is no mechanism for recovery of the top glass slide which is often subsequently lost, often dropping into the bottom of the insertion lock chamber from where recovery may be extremely difficult to achieve. Once removed from the cold stage, the portion of the sample still attached to this slide will then start to increase in temperature and outgas which can compromise the instrument vacuum. Further problems such as the fracture probe misalignment resulting in failure to fracture the sample or even the loss of both slides frequently occur. Additionally, contact with the probe can cause temperature variations and any failure to maintain the whole sample assembly uniformly at low temperature can result in spoilage of the sample. Finally, there is no guarantee that the lower slide will contain a surface suitable for analysis as the sample may fracture along the interface with the lower slide and the majority of the sample may remain on the top slide which is then lost in the vacuum chamber.

Any solution to these problems should be effective to provide a clean surface with low outgassing suitable for surface analysis, reliable, robust and be easy to implement by the end user.

The current invention overcomes all these limitations in both a novel and cost effective manner.

According to the present invention, the sample fracture is achieved by an automated mechanism built into the sample holder instead of using a manual method similar to those described above. The invention completely removes the probability of losing the sample, completely removes the problem of loss of the top slide by allowing it to be retained and available for analysis thereby removing the problem of which slide the sample adheres to. It also significantly improves control of the temperature of the whole sample and slide assembly and, through simpler and easier handling, reduces the possibility of frosting of the sample or sample slide during transfer into the vacuum.

DESCRIPTION OF FIGURES

An apparatus for the preparation and presentation of fluid bearing materials for surface analysis in a vacuum and accompanying method are now described by way of example of embodiments only and by reference to the accompanying drawings, of which

FIG. 1 shows an isometric view of an example of a sample slide assembly

FIG. 2 shows an isometric view of an example of a sample slide and sample holder assembly with operating mechanism

FIG. 3 shows a cross section view of an example of a sample slide and sample holder assembly with operating mechanism

FIG. 4 shows a schematic of the operation of the plates.

DETAILED DESCRIPTION

A preferred embodiment of the invention as related to the presentation of clean surfaces from biological sample such as tissue for SIMS analysis is shown in FIGS. 1 to 4.

FIG. 1 shows a replacement for the two slides that were described above and FIG. 4 a schematic of the operation of the plates. This comprises of two stainless steel metal plates (1) and (2) joined together by a hinge pin (3) said plates being so disposed as to allow rotation with respect to each other around the hinge pin (3). Alternatively the slide plates may be constructed from any suitable material such as any metal, glass, silicon or other material or combination thereof. The two plates and the pin form a floating hinge assembly whereby the plates can accommodate samples of varying thickness and of different thicknesses. The sample (44) would typically be a drop of liquid suspension or a piece of biological tissue which is placed on the surface of the lower plate (1) as shown in FIG. 4 a with the upper plate opened so that the upper side of the lower plate is accessible to mount the sample. The upper plate (2) is then rotated around the hinge pin (3) to be approximately facing the lower plate and the sample (44) is trapped between the lower side of the upper sample plate and the upper side of the lower sample plate as shown schematically in FIG. 4 b. If required, a suitable spacer arrangement may be used to maintain a minimum sample thickness. At this point, if required, pressure to the plates may be applied to give, for example, exclusion of air and to ensure good contact of the sample with both plates. The hinge assembly, complete with trapped sample, is then transferred to a cryogenic bath (often liquid Nitrogen (LN2) or a “slush” of ice and LN2) which already contains the sample holder assembly. Once the sample has frozen completely the upper plate should be bonded to the lower plate by the frozen sample. Whilst still keeping both the hinge assembly and the sample plate assemblage submerged in the coolant, the lower plate of the hinge is clamped to the holder using screws or clips. This procedure can either take place within a dry atmosphere glove box or, if outside of the dry atmosphere, the assemblage is kept cooled in the cryogenic bath while the bath is transferred to a dry atmosphere glove box which is mounted around the entry lock of the vacuum instrument. Alternative procedures involving more rapid freezing and multiple cryogenic baths with different cooling properties (typically used to increase the speed of freezing to avoid crystalline structure formation in the sample fluid) are available and may be used provided the sample slide is kept optimally cooled during the procedure and the mounting of the sample to the sample holder is done under cryogenic conditions and the whole assembly is kept optimally cooled until final transfer under a dry gas environment into the vacuum.

The assembly of hinge and sample holder is shown in isometric view in FIG. 2 and in section in FIG. 3. The sample holder (3) is a heat-conductive metal block. A spring-loaded flipper bar (7) is mounted on a pin (6) and a release lever (1) retains this flipper bar.

The fracture mechanism is set and the complete sample holder and hinge assembly is then promptly transferred from the cryogenic bath to the insertion lock of the instrument where it is placed on cold stage and the insertion lock is pumped down to UHV. A transfer probe is used to engage the sample and move it to the analysis chamber where it is mounted on a cryogenically cooled stage. In the process of engaging the sample, the probe is used to trigger the fracture mechanism. The probe, which can exert significant force, presses against the protrusion on the release lever (1), the release lever pivots and the spigot (8) pushes upward on the top plate (2) of the hinged sample plates at point (A). This causes the sample (44) to fracture in a plane approximately parallel to the hinged plates. The release lever pivots further and releases the flipper bar (7). This flipper bar automatically opens the hinge to a preferred angle of 180 degrees, leaving both halves (45 & 46) of the sample (44) facing up and available for analysis as shown schematically in FIG. 4 c. The spring and flipper bar also retains the upper plate firmly against the sample holder. 

1. An apparatus for the freeze-fracture and presentation for surface analysis in a vacuum of clean surfaces from frozen fluid containing materials, said apparatus incorporating; a pair of sample plates joined by a hinge pin to form a floating hinge; and said plates being so disposed as to be able to face each other and also to be capable of rotating around said hinge pin with respect to each other; and a means of support for the said sample plates suitable for insertion into a vacuum system; and a means by which said sample plates can be mechanically prised apart in the vacuum; and a means of opening the said hinged sample plates such that they rotate with respect to each other through a predefined rotation angle; and a means of releasing said opening means inside the vacuum.
 2. An apparatus as claimed in claim 1 where the said means of mechanically prising the sample plates apart in vacuum is an externally operated mechanical device.
 3. An apparatus as claimed in claim 1 where the said means of prising the sample plates in vacuum is an electrical or electromagnetic device.
 4. An apparatus as claimed in claim 1 where the said means of mechanically prising the sample plates apart in vacuum is the transfer probe.
 5. An apparatus as claimed in claim 1 where the said means of opening said sample plates is a spring loaded bar. 6-7. (canceled)
 8. An apparatus as claimed in claim 1 where the said hinged sample plates and said sample holder are so arranged that after being prised apart and opened, the said sample plates lie at a predefined angle between 90 and 270 degrees with respect to each other.
 9. An apparatus as claimed in claim 1; where the said hinged sample plates and sample holder are so arranged that after being prised apart, the said sample plates lie substantially in the same plane with both recently fractured sample surfaces facing substantially in the same direction.
 10. (canceled)
 11. An apparatus as claimed in and of claim 1 where a single lever operates the fracture action and the release of the sprung action for opening the hinged plates.
 12. An apparatus as claimed in claim 1 where the fracture and hinge opening mechanisms are both actuated by a single mechanical drive by either manual or automatic means.
 13. An apparatus as claimed in claim 1 where all the materials of the said sample slides and said sample holder have a high thermal conductivity.
 14. An apparatus as claimed in claim 1 where all the materials of the said sample slides and said sample holder are suitable for use in ultra high vacuum.
 15. (canceled)
 16. An apparatus as claimed in claim 1 where samples of varying thicknesses between 0.001 mm and 2.5 mm can be accommodated between the sample plates.
 17. An apparatus as claimed in claim 1 where the sample is contained within a well or indent formed on either sample plate.
 18. An apparatus as claimed in claim 1 where multiple samples can be accommodated between the sample plates.
 19. An apparatus as claimed in claim 1 where biological samples can be mounted for analysis in a SIMS instrument.
 20. An apparatus as claimed in claim 1 where biological samples can be mounted for analysis in a surface analysis instrument.
 21. An apparatus as claimed in claim 1 where the sample may be any organic sample.
 22. (canceled)
 23. A method for presentation of a frozen sample with minimised surface contamination and frosting for analysis using a surface analysis techniques comprising the steps of; placing a sample between a pair of hinged sample plates comprising an upper plate and a lower plate; and applying pressure if required to ensure a good contact between the sample plates and the sample; and rapidly freezing the sample plates containing the sample between them in a cryogenic bath in a dry gas environment: and mounting the lower sample plate to a sample mount that is already immersed in the cryogenic bath by means of screws or clamps; and setting the fracture mechanism; and whilst still in the dry gas environment transferring the sample plate and sample mount assemblage to a cryogenically cooled stage in an insertion lock and placing it promptly under ultra high vacuum; and freeze-cleaving the sample by mechanical means to separate the sample slides and opening them such that both the freeze-cleaved surfaces are facing in a direction suitable for surface analysis; and transferring the sample holder assemblage to a second cryogenically cooled stage in the analysis position.
 24. A method for presentation of a frozen sample as claimed in claim 23 where the rapidly freezing of the sample plates containing the sample between them in a cryogenic bath takes place outside of a dry gas environment and whilst the assemblage is still cooled in the cryogenic bath transferring them both to a dry gas environment.
 25. A method for presentation of a frozen sample as claimed in claim 23 where the sample is any biological or biochemical material requiring freezing for surface analysis. 26-27. (canceled) 